Formulation for iron chelation therapy and method of preparation thereof

Abstract

The present disclosure relates to a formulation for iron chelation therapy comprising: a water-soluble polyphenol-biogenic amine complex comprising of a polyphenol and an amine, and a 0.9% of sodium chloride; optionally one or more additive, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination. The present disclosure also provides a method of preparation of a formulation for iron chelation therapy comprising: a) adding 0.005 to 5% w / w of a polyphenol and 0.005 to 5% w / w of an amine followed by adjusting to pH-7 by adding HCl / NaHCO3 to form a mixture; and b) mixing the mixture of step a) with suitable proportion in 0.9% sodium chloride and optionally one or more additive to form a formulation for iron chelation therapy, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

Description

FORMULATION FOR IRON CHELATION THERAPY AND METHODOF PREPARATION THEREOFFIELD OF THE INVENTION

[0001] The present disclosure relates to a field of pharmaceutical formulation. Particularly, the present disclosure relates to a formulation for iron chelation therapy. The present disclosure also provides a method of preparation of a formulation for iron chelation therapy.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Iron Chelation Therapy (ICT) is a medical treatment used to treat excess iron content in the body. This condition is caused by a variety of factors such as frequent blood transfusions, genetic illnesses such as thalassemia, and other medical reasons. Iron overload can cause significant health issues, causing damage to the key organs, including the liver, spleen, and kidney. ICT employs chelating agents, which are molecules that bind to excess iron and enable its elimination from the body. The potent synthetic drugs used in ICT are Deferoxamine (DFO), Deferiprone and Deferasirox, etc. However, recent studies have examined the potential of natural and endogenous compounds as iron chelators.

[0004] CA is a naturally occurring phenolic chemical found in plants such as coffee, fruits, and vegetables. It has a variety of biological actions, including anti-oxidant, anti- inflammatory, and anti-bacterial properties. The catechol unit present in CA is known to have high affinity and selectivity towards iron. However, one drawback of CA is its low water solubility.

[0005] ICT tackles iron overload by binding and removing excess iron. Previous approaches, such as phlebotomy and early chelators like deferoxamine, had considerable downsides, including difficulty in administration and adverse effects. Modem chelation therapy, with enhanced oral agents and combination techniques, provides more effective and patient- friendly alternatives, yet issues such as side effects and expense persist. Ongoing research attempts to improve these medicine's safety, efficacy, and patient adherence.

[0006] While encouraging, more studies are needed to realize the full potential of natural chelators in clinical practice. Integrating these substances into therapy regimens allowshealthcare providers to deliver more comprehensive and patient-friendly methods for iron overload management.

[0007] Several studies have investigated various polyphenols’ anti-bacterial, anti-oxidant, and anti-cancer activities, including CA, Ferulic acid, etc., and their synthetic counterparts. However, most natural polyphenols have a considerable limitation in terms of water solubility. Furthermore, synthetic derivatives such as amides and esters, which require covalent changes at the carboxylic acid end, frequently suffer from reduced water solubility. Additionally, due to the permanent change in the chemical structure, their innate properties could have been altered in the biological system.

[0008] It was evident from the literature that DFO is the gold-standard in ICT [Tamagno et al., Asian J. Transfus. Sci., 2022, 11(1), 13-17], Other compounds like ferrostatin-1, liproxstatin-1, and vitamin E are also used towards this one [Mu et al., Sci. Bull., 2021, 66 (17), 1806-1816], Researchers have also studied controlled delivery of DFO by incorporating them into a chitosan-based nanosponge [Lv et al., Adv. Sci., 2024, p2305895], DFO conjugated with a nanochelator, s-poly-L-lysinc. was demonstrated to be highly efficacious in renal clearance of excess iron in a mice model [Kang et al., Nat. Comm., 2019, 10(1), p.5134], Polyamine-hydroxypyridinone conjugate has been shown to be potential chelators in in vitro conditions, but no in vivo studies using these compounds are available [Bergeron et al., J. Med. Chem., 2003, 46(25), 5478-5483],

[0009] Alikhani et al. [E. J. Pharmacol., 2022, 924, p.951725] have studied the efficacy of several natural compounds such as caffeic acid, ferulic acid, coumarin, etc., to treat iron overload. However, it was not using acid-base complexes of the catechol-containing compounds and thus different from the present invention. Furthermore, CA has inherent poor water solubility, and thus without enhancing its water solubility, it is likely that CA was administered within its solubility limit. The same group later reported the beneficial effects of CA and ferulic acid for ICT. In this case, the authors mentioned the use of 800 pg / mL of concentration for the in vitro study. For the in vivo study, the CA dosage was varied from 30 mg / kg / day to 50 mg / kg / day. However, for such a dosage, a huge volume of aqueous phase has to be used in order to tackle the poor water solubility of CA. For instance, 30 mg / kg / day would require approximately 30 mb of aqueous phase to be used per kg, which is not the case with the acid-base complexes developed of the present invention that show highly enhanced water solubility.

[0010] A study by Hawula et al. [Biometals, 2021, 34(4), 855-866] reported the in vitro screening of several catechol-containing chelators as potential ICT therapeutic agents from the Davis open access natural product-based library. About 9 compounds were found to be promising, out of which one compound, RAD362 [N-(3-aminopropyl)-3,4- dihydroxybenzamide, was found to be promising in protecting against DNA damage in the hepatocyte and breast cancer cell lines. However, no in vivo study on these compounds were found. Shao et al., [Antioxidants, 2021, 10(5), 798] reported in vitro studies showing high capacity of caffeic acid phenyl ester (CAPE) due to its complexation ability with iron. However, no in vivo studies are reported in this work. Also, the aqueous based formulations of such compounds are also unclear due to their poor water solubility. Further, such compounds do not belong to the class of catechol-containing acid-base complexes, as presented in the present invention.

[0011] Marzabadi et al., [Free Radical Biology and Medicine, Vol. 21, Issue 3, 1996, Pages 375-381] reported that spermine (a biogenic amine) helps to prevent iron accumulation in an in vitro study.

[0012] Thus, to the best of inventor’s knowledge, no report was identified describing the utility of water-soluble CA-biogenic amine derivatives for treating iron overload conditions.OBJECTIVES OF THE INVENTION

[0013] An objective of the present disclosure is to provide a formulation for iron chelation therapy.

[0014] Another objective of the present disclosure is to provide a method of preparation of a formulation for iron chelation therapy.

[0015] Still another objective of the present disclosure is to develop a novel, non-cytotoxic, cost-effective and highly biocompatible formulation for iron chelation therapy.

[0016] Yet another objective of the present disclosure is to reduce the body's iron burden, prevent iron-induced tissue damage, and enhance overall patient outcomes.SUMMARY OF THE INVENTION

[0017] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0018] An aspect of the present disclosure provides a formulation for iron chelation therapy comprising: formulation for iron chelation therapy comprising: a water-soluble polyphenolbiogenic amine complex comprising of a polyphenol and an amine, and a 0.9% of sodium chloride; optionally one or more additive, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

[0019] Another aspect of the present disclosure provides a method of preparation of a formulation for iron chelation therapy comprising: a) adding 0.005 to 5% w / w of a polyphenol and 0.005 to 5% w / w of an amine followed by adjusting to pH-7 by adding HCI / NaHCO? to form a mixture; and b) mixing the mixture of step a) with suitable proportion in 0.9% sodium chloride and optionally one or more additive to form a formulation for iron chelation therapy, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

[0020] Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the exemplary embodiments of the invention.DESCRIPTION OF THE FIGURES

[0021] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0022] Figure 1 illustrates experimental flow.

[0023] Figure 2 CA-Spermine and Histidine combinations rescues HEK-293 cells from Irondextran induced toxicity. (A) Effect of CA-Spermine combination in Iron-Dextran toxicity, and (B) Effect of CA-Histidine combination in Iron-dextran toxicity.

[0024] Figure 3 illustrates biocompatibility of (A) CA-Spermine and (B) CA-Histidine combinations.

[0025] Figure 4 illustrates CA-Sp and CA-His combinations reduced the levels of proinflammatory marker IL-6 induced by iron overload.

[0026] Figure 5 illustrates CA-Sp and CA-His combinations on reduced the levels of Iron toxicity marker Ferritin induced by iron overload.

[0027] Figure 6 illustrates CA-Sp and CA-His combinations reduced the levels of pro- inflammatory marker TNF-a induced by iron overload.

[0028] Figure 7 illustrates treatment with CA-Sp and CA-His combinations reduced iron depositions in major organs negating Iron dextran induced toxicity.

[0029] Figure 8 illustrates Treatment with CA-Sp and CA-His combinations reduced iron- induced organ toxicities negating effects due to Iron dextran: (A) Creatinine, (B) AST, and (C) ALT levels in the serum.DETAILED DESCRIPTION OF THE INVENTION

[0030] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0031] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0032] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0033] In some embodiments, numbers have been used for quantifying weights, percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numericalranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0034] The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0035] Unless the context requires otherwise, throughout the specification which follows, the word “comprise” and variations thereof, such as “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”

[0036] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

[0037] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Furthermore, the ranges defined throughout the specification include the end values as well, i.e., a range of 1 to 10 implies that both 1 and 10 are included in the range. For the avoidance of doubt, the applicant shall be entitled to any equivalents according to applicable law.

[0038] All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0039] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.

[0040] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0041] It should also be appreciated that the present disclosure can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0042] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0043] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0044] The term “or”, as used herein, is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0045] Various terms are used herein to the extent a term used is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0046] The present disclosure provides a formulation that reduces the body's iron burden, prevent iron-induced tissue damage, and enhance overall patient outcomes. This is accomplished using chelating agents, which bind to excess iron and help it excrete from the body. Standard iron chelators like Deferoxamine help selectively bind to ferric ion (Fe3+) and create complexes that are later eliminated through urine and faeces, helping maintain healthy iron levels and reducing the hazards associated with iron overload. Since there are limitations even with the usage of these standard compounds, the water-soluble compounds CA-Sp and CA-His of the present disclosure aid in chelating the excess iron present in the body and prove to be promising candidates in the iron chelation therapy, thereby benefiting the pharmaceutical sector.

[0047] Taking lead from the known fact that catechol units have high affinity towards iron, the present invention employed water soluble CA-biogenic amine acid-base complexes as potential ICT therapeutic agents. This strategy enables one to completely utilize natural products that are highly biocompatible. Therefore, to demonstrate the proof-of-concept of employing catechol-containing naturally occurring polyphenols for ICT application, CA-Sp and CA-His are selected as the model compounds. Through simple intraperitoneal injection, the formulated drugs were found to be highly efficacious to a similar tune to that of the clinical gold-standard drug, Deferoxamine. The in vivo study additionally revealed the noncytotoxicity of the formulated drugs and thereby, proved as high potential ICT agents.

[0048] Due to their extreme facile nature of formulation, the multi-step organic synthesis, release of hazardous by-products, utilization of harmful organic solvents and purification steps are completely avoided. Hence, the formulated drugs are not only highly biocompatible but also cost-effective and eco-friendly.

[0049] An embodiment of the present disclosure provides a formulation for iron chelation therapy comprising: a water-soluble polyphenol-biogenic amine complex comprising of a polyphenol and an amine, and a 0.9% of sodium chloride; optionally one or more additive, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

[0050] In an embodiment, the water-soluble polyphenol-biogenic amine complex is present in the formulation in an amount in the range of 0.01 to 10%w / w, preferably 0.1-5.0%w / w, more preferably, 0.1-3.0%w / w.

[0051] In an embodiment, the polyphenol is selected from a group consisting of caffeic acid, tannic acid, gallic acid, ferulic acid and combination thereof. Preferably, the polyphenol is caffeic acid. The polyphenol is present in an amount in the range of 0.005 to 5%w / w, preferably 0.05-3%w / w, more preferably 0.05-1.5%w / w.

[0052] In an embodiment, the amine is selected from a group consisting of spermine, histidine, spermidine, arginine, dopamine and combination thereof. Preferably, the amine is selected from spermine and histidine. The amine is present in an amount in the range of 0.005 to 5%w / w, preferably, 0.05-3%w / w, more preferably, 0.05-1.5%w / w.

[0053] In an embodiment, the additive is selected from a group consisting of thickening agent, bulking agent, lubricant, solubilizer, binder, colouring agent, tonicity agent, buffering agent, preservative, moisturizing agent, fdm former / waterproofmg agent, rheology modifying agent, fragrance, emollient, humectants, emulsifier, diluents, solvent and combinationthereof. The additive is present in the formulation in an amount in the range of 0.01 to 10 % w / w.

[0054] In an embodiment, the thickening agent(s) includes but not limited to, carbopol, carbomer, acrylate copolymer, beeswax, emulsifying cresmer wax, sodium acrylate / sodium acryloyl dimethyl tauratecopolymer, and combinations thereof. However, a person skilled in the art would appreciate that any other thickening agent(s) can be utilized to serve the intended purpose.

[0055] In an embodiment, the bulking agent(s) include but not limited to, lactose USP, starch 1500, mannitol, sorbitol, maltodextrin, maltitol or other non-reducing sugars; microcrystalline cellulose (e.g., Avicel), dibasic calcium phosphate (anhydrous or dihydrate), sucrose, and combinations thereof. However, a person skilled in the art would appreciate that any other bulking agent(s) can be utilized to serve the intended purpose.

[0056] In an embodiment, the lubricant(s) includes but not limited to, zinc stearate, magnesium stearate, stearic acid, calcium stearate, Vegetable stearin, and combinations thereof. However, a person skilled in the art would appreciate that any other lubricant(s) can be utilized to serve the intended purpose.

[0057] In an embodiment, the solubilizer(s) includes but not limited to, cyclodextrins, pH adjusters, salts and buffers, surfactants, fatty acids, phospholipids, metals of fatty acids, and combinations thereof. However, a person skilled in the art would appreciate that any other solubilizer(s) can be utilized to serve the intended purpose.

[0058] In an embodiment, the binder(s) include but not limited to, cellulosic derivatives (such as methylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropylmethyl cellulose etc), polyacrylates (such as Carbopol, polycarbophil, etc), Povidone (all grades), Polyox of any molecular weight or grade, irradiated or not, maize starch, povidone, copovidone, com starch, starch, polyvinylpyrrolidone (PVP), microcrystalline cellulose (Avicel@ -Avicel 101), and combinations thereof. However, a person skilled in the art would appreciate that any other binder(s) can be utilized to serve the intended purpose.

[0059] In an embodiment, the colouring agent(s) includes but not limited to, El 02 Tartrazine, E104 Quinoline Yellow, El 10 Sunset Yellow FCF, E120 - Cochineal, carminic acid, Carmines, E122 Azorubine (Carmoisine), E123 Amaranth, E124 Ponceau 4R (Cochineal Red A), E127 Erythrosine, E129 Allura Red, E131 Patent Blue, and combinations thereof. However, a person skilled in the art would appreciate that any other colouring agent(s) can be utilized to serve the intended purpose.

[0060] In an embodiment, the tonicity agent(s) includes but not limited to, dextrose, glycerin, mannitol, potassium chloride, sodium chloride, and combinations thereof. However, a person skilled in the art would appreciate that any other tonicity agent(s) can be utilized to serve the intended purpose.

[0061] In an embodiment, the buffering agent(s) includes but not limited to, sodium citrate, potassium citrate, sodium citrate di-hydrate, citric acid, citric acid monohydrate, sodium bicarbonate, potassium bicarbonate, sodium di-hydrogen phosphate and potassium dihydrogen phosphate, and combinations thereof. However, a person skilled in the art would appreciate that any other buffering agent(s) can be utilized to serve the intended purpose.

[0062] In an embodiment, the preservative(s) includes but not limited to, sodium benzoate, potassium sorbate, phenoxyethanol, p-hydroxybenzoic acid esters, sorbic acid, benzoic acid, propionic acid or salts thereof; Alcohols such as benzyl alcohol, butanol or ethanol, isopropyl alcohol and quaternary ammonium compounds such as benzalkonium chloride, sodium benzoate, and combinations thereof. However, a person skilled in the art would appreciate that any other preservative(s) can be utilized to serve the intended purpose.

[0063] In an embodiment, the moisturizing agent(s) includes, but are not limited to lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, butylene glycol, sodium PCA, Carbowax 200, Carbowax 400, Carbowax 800, and combinations thereof. However, a person skilled in the art would appreciate that any other moisturizing agent(s) can be utilized to serve the intended purpose.

[0064] In an embodiment, the film former / waterproofmg agent(s) includes, but are not limited to acrylates / C 12-22 alkylmethacrylate copolymer, C30-38 olefm / isopropyl maleate / methylacrylate copolymer, polyethylene, waxes, vinylpyrrolidone / dimethiconylacrylate / polycarbamylpolyglycol ester, butylated polyvinylpyrrolidone, polyvinylpyrrolidone / hexadecane copolymer, polyvinylpyrrolidone / eicosene copolymer, tricontanylpolyvinylpyrrolidone, Brassica campestris / Aleuritisfordi Oil copolymer, aminofunctional silicones, decamethylcyclopentasiloxane (and) trimethylsiloxysilicate, octadecene / methylacrylate copolymer, and combinations thereof. However, a person skilled in the art would appreciate that any other fdm former / waterproofmg agent(s) can be utilized to serve the intended purpose.

[0065] In an embodiment, the rheology modifying agent(s) include, but are not limited to acrylates crosspolymer, acrylates / C 10-30 alkylacrylate crosspolymer, polyacrylic acid, sodium polyacrylate, polyacrylate, acrylate s / vinyl ester copolymer, PVP / decene copolymer,styrene / MA copolymer, acetamide MEA, acrylamides copolymer, acrylates copolymer, acrylates / steareth-20 methacrylate copolymer, acrylates / beheneth-25 methacrylate copolymer, PEG-150 / decyl alcohol / SMDI copolymer, PVP, PVM / MA decadiene crosspolymer, carbomer, PEG crosspolymer, acrylates / palmeth-25 acrylates copolymer, polysaccharide, polyether- 1, sodium magnesium silicate, bentonite, trihydroxystearin, hydroxy stearate, aluminum-magnesium hydroxide stearate, acacia gum, xanthan gum, microcrystalline cellulose, cellulose gum, and combinations thereof. However, a person skilled in the art would appreciate that any other rheology modifying agent(s) can be utilized to serve the intended purpose.

[0066] In an embodiment, the fragrance(s) includes, but are not limited to alcohols, aldehydes, ketones, esters, ethers, acetates, nitriles, terpene hydrocarbons, nitrogenous or sulfurous heterocyclic compounds and essential oils. However, naturally occurring plant and animal oils and exudates comprising complex mixtures of various chemical components are also know for use as fragrance materials. The individual perfume raw materials which comprise a known natural oil can be found by reference to Journals commonly used by those skilled in the art such as Perfume and Flavourist; or Journal of Essential Oil Research, or those listed in reference texts such as the book by S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA and re-published by Allured Publishing Corporation Illinois (1994). Additionally, some perfume raw materials are supplied by the fragrance houses (Firmenich, International Flavors & Fragrances, Givaudan, Symrise) as mixtures in the form of proprietary 14 speciality accords. Non-limiting examples of the fragrance materials useful herein include pro- fragrances such as acetal pro-fragrances, ketal pro-fragrances, ester pro-fragrances, hydrolyzable inorganic-organic pro-fragrances, and combinations thereof. However, a person skilled in the art would appreciate that any other fragrance(s) can be utilized to serve the intended purpose.

[0067] In an embodiment, the emollient(s) includes but not limited to, caprylic / caprictriglyceride (e.g., Endimulse 33V), isononylisononanoate, squalane (e.g., neossancesqualane), patauaoil (e.g, oenocarpusbataua fruit oil), and combinations thereof. However, a person skilled in the art would appreciate that any other emollient(s) can be utilized to serve the intended purpose.

[0068] In an embodiment, the humectant(s) includes but not limited to, Glycerine, Propylene Glycol, Butylene glycol, aloe-vera gel, hexylene glycol, 25 glyceryl triacetate, Sodium hyaluronate, and combinations thereof. However, a person skilled in the art would appreciate that any other humectant(s) can be utilized to serve the intended purpose.

[0069] In an embodiment, the emulsifier(s) includes but not limited to, hydrogenated lecithin, C12-16 Alcohols, palmitic acid (e.g., Biophillic™ H) and cetylalcohol, glyceryl stearate, PEG-75 stearate, ceteth-20, steareth-2 (Emulium® Delta), and combinations thereof. However, a person skilled in the art would appreciate that any other emulsifier (s) can be utilized to serve the intended purpose.

[0070] In an embodiment, the diluent(s) includes but not limited to, microfine cellulose, lactose, starch, pregelatinized starch, calcium carbonate, calcium sulfate, sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, potassium chloride, powdered cellulose, sodium chloride, sorbitol, talc, and combinations thereof. However, a person skilled in the art would appreciate that any other diluent(s) can be utilized to serve the intended purpose.

[0071] In an embodiment, solvent(s) includes but not limited to, methanol, ethanol, n- propanol, isopropanol, hexane, heptane, petroleum ether, cyclohexane, diethyl ether, diisopropyl ether, ethyl acetate, methyl acetate, ethyl formate, methyl formate, isobutyl acetate, n-butyl acetate, methylene chloride, ethylene chloride, chloroform, carbon tetrachloride, acetone, ethyl methyl ketone, diisobutyl ketone, methyl isobutyl ketone, 1,4- dioxane, toluene, ammonia solution, glacial acetic acid, ammonium hydroxide, sodium hydroxide, calcium hydroxide, calcium carbonate, potassium hydroxide, potassium carbonate, water, and combinations thereof. However, a person skilled in the art would appreciate that any other solvent(s) or a combination of solvent(s) can be utilized to serve the intended purpose.

[0072] In an embodiment, the formulation is administered by parentally with a dose in the range of 1-500 mg / kg / day. Preferably, the formulation is administered by intraperitoneal injection. Preferably, the dose is 10-100 mg / kg / day.

[0073] Another embodiment of the present disclosure is to provide a method of preparation of a formulation for iron chelation therapy comprising: a) adding 0.005 to 5% w / w of a polyphenol and 0.005 to 5% w / w of an amine followed by adjusting to pH-7 by adding HCI / NaHCCf to form a mixture; and b) mixing the mixture of step a) with suitable proportion in 0.9% sodium chloride and optionally one or more additive to form a formulation for iron chelation therapy, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination. The process of CA- Sp / CA-His complex formulation is given in Scheme 1.Mix the components in suitable proportion in 0.9% Nad

[0074] Scheme 1 illustrates a preparation of CA-Sp / CA-His complex formulation.

[0075] The present disclosure initially developed an iron overload mice model by parenterally administering iron dextran solution intraperitoneally. Once the mice model was developed, a standard drug Deferoxamine mesylate, and complexes of CA with natural polyamines like Sp and His were administered as ICT therapeutic agents.

[0076] Figure 1 shows experimental flow. Iron overload was induced by intraperitoneal injection (IP) of Iron dextran (ID) (150 mg / kg) followed by treatment with standard Deferoxamine (DFO) (100 mg / kg, IP) or investigational compounds like CA-Sp (8.8 + 10 mg / kg) and CA-His (50 + 42.8 mg / kg, IP). Ferritin, pro-inflammatory markers like IL-6 and TNF-a along with organ toxicity markers like ALT (Alanine transaminase), AST (Aspartate transaminase), and Creatinine were estimated in serum from various animal groups. At the end of the study, animals were sacrificed. Liver, kidney and Spleen were collected, sectioned and stained with Prussian blue to estimate Iron levels in various organs. The levels of all the markers were compared with untreated (control) and Iron overloaded animals (ID group) to evaluate the efficacy of the therapy.

[0077] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.EXAMPLES

[0078] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.Example 1: CA-Sp complex formulation

[0079] About 0.88 mg of CA and 1 mg of Sp were added in 997 pL of 0.9% w / w NaCl followed by adjusting to pH-7 by adding 3 pL of 12 M HC1 to form a formulation for iron chelation therapy.Example 2: CA-His complex formulation

[0080] About 5 mg of CA and 4.28 mg of His were added in 945 pL of 0.9% w / w NaCl followed by adjusting to pH-7 by adding 55 pL of 1 M NaHCCf to form a formulation for iron chelation therapy.Example 3: in vitro for the cytotoxicity of CA-Sp and CA-His

[0081] The invention was tested in vitro for the cytotoxicity of CA-Sp and CA-His with the Human Embryonic Kidney (HEK) cells. These compounds were also studied in combination with iron dextran (ID), which was used for inducing iron overload in the in vivo mice model.

[0082] Figure 2 showed CA-Spermine and Histidine combinations rescues HEK-293 cells from Iron-dextran induced toxicity. Figure 2 (A) showed the effect of CA-Spermine combination in Iron-Dextran toxicity, and Figure 2 (B) showed the effect of CA-Histidine combination in Iron-dextran toxicity. HEK-293 cells were exposed to the IC50 concentration of Iron dextran to mimic Iron toxicity. Iron dextran exposed cells were treated with increasing concentrations of CA-Spermine and Histidine combinations. Low concentrations of CA-Spermine and CA-Histidine (0.39 & 0.78 mM) combinations were effective in negating Iron-dextran-induced toxicity in HEK-293 cells. Among these two, 0.78 mM concentration was considered for carrying out further in-vivo studies.

[0083] From the in-vitro MTT assay, where initially HEK-293 cells were subjected to the IC50 concentration of Iron dextran (15 mM) to simulate iron toxicity in the in-vitro model, were evaluated with increasing doses of CA-Spermine and Histidine combinations. Among the tested concentrations, the results revealed that higher concentrations of more than 0.78 mM showed a decrease in the viability profile which indicates that these concentrations are ineffective to the HEK cells. Both 0.39 and 0.78 mM concentrations of the CA-Spermine and CA-Histidine combinations effectively counteracted the Iron-dextran-induced toxicity in the HEK-293 cells and exhibited more than 50% cell viability. 0.78 mM concentration was foundto be the best when compared to 0.39 mM, thus this concentration was considered for the in- vivo studies for further analysis.

[0084] Biocompatibility of (A) CA-Spermine and (B) CA-Histidine combinations as shown in Figure 3. The biocompatibility studies unveiled that both the concentrations of CA- Spermine and CA-Histidine combinations (0.39 and 0.78 mM) were found to be non-toxic to the HEK-293 cells which suggests that these compounds are safe for utilization. Higher concentrations beyond 0.78 mM were noticed to be toxic and incompatible, while on the contrary, 0.78 mM concentration was selected for further in-vivo studies, which was likely due to its robust therapeutic effect.Example 4: in vivo efficacy in a mice model

[0085] The invention was then tested for the in vivo efficacy in a mice model by employing representative organic acid-base complexes by choosing CA-Sp and CA-His as the constituents, and the results are compared with DFO as the gold-standard drug. Fe-dextran (150 mg / kg mice) was administered on Day 1 through the intraperitoneal route in all the 14- 16 weeks old BALB / c mice, excluding the control group, and was left for a week. After creating the iron overload, the subjects from different groups were administered with DFO (100 mg / kg), CA-Sp (CA: 8.8 mg / kg and Sp: 10 mg / kg), and CA-His (CA: 50 mg / kg and His: 42.8 mg / kg) on 8th, 11th, 14th, and 17thdays, and the subjects were monitored till the 21stday. Following the termination of the animals on the 21stday, the blood and serum samples were collected and the IL-6, Ferritin, and TNF-a levels were monitored by Sandwich ELISA.

[0086] The biochemical analysis was also done by checking the serum creatinine, AST, and ALT levels in all the groups of mice such as control (no iron overload), negative control (ID), positive control (DFO), and test groups (CA-Sp and CA-His). The histopathological staining of the organs like kidney, liver, and spleen was carried out by the Prussian blue stain method for all the groups. These tests confirmed that the formulations are highly potent to chelate the excess iron effectively and thereby restore the normal physiological conditions in the animals.

[0087] Figure 4 showed CA-Sp and CA-His combinations reduced the levels of proinflammatory marker IL-6 induced by iron overload. ID injection led to an increase in IL- 6, which was attenuated by standard (DFO) and investigational (CA-Sp and CA-His) treatments.

[0088] The IL-6-specified ELISA study with the mice serum samples demonstrated that there was an observed increase of IL-6 levels in the Iron overload group which was consistent withthe well-established inflammatory response associated with excess iron, which can lead to oxidative stress and activate pro-inflammatory pathways. The reduction of IL-6 levels by both the standard treatment (Deferoxamine mesylate, DFO) and the investigational treatments (CA-Sp and CA-His) suggests that these compounds possess anti-inflammatory properties and alleviate iron-induced oxidative stress. This signifies that these compounds could serve as viable adjuncts to existing therapies for iron overload. Moreover, their ability to modulate inflammatory responses highlights their potential therapeutic application not only in iron overload conditions but also in other diseases driven by oxidative stress and inflammation.

[0089] Figure 5 showed CA-Sp and CA-His combinations on reduced the levels of Iron toxicity marker Ferritin induced by iron overload. ID injection led to an increase in serum ferritin, which was attenuated by standard (DFO) and investigational (CA-Sp and CA-His) treatments.

[0090] Elevated Serum Ferritin is a common indicator of excessive iron accumulation in the body, reflecting increased iron storage and often correlating with oxidative damage and inflammation. Ferritin ELISA stated that there was attenuation of serum ferritin levels by both the standard treatment (Deferoxamine, DFO) and the investigational treatments (CA-Sp and CA-His) suggesting that these compounds are effective in mitigating iron overload. This implies that these compounds chelate iron and reduce iron-induced stress, leading to the decrease in ferritin levels. Moreover, it was observed that the investigational treatments with CA-Sp and CA-His were much better compared to the commercial standard DFO.

[0091] Figure 6 showed CA-Sp and CA-His combinations reduced the levels of pro- inflammatory marker TNF-a induced by iron overload. ID injection led to an increase in TNF-a, which was attenuated by standard (DFO) and investigational (CA-Sp and CA-His) treatments.

[0092] Elevated TNF-a levels are commonly associated with oxidative stress and inflammation, both of which are exacerbated by iron overload. The results of TNF-a ELISA revealed the ability of CA-Sp and CA-His treatments to lower TNF-a levels, similar to the standard treatment DFO, suggesting that CA-Sp and CA-His possess anti-inflammatory properties in addition to their potential for mitigating iron toxicity.

[0093] Figure 7 showed Treatment with CA-Sp and CA-His combinations reduced iron depositions in major organs negating Iron dextran induced toxicity.

[0094] Iron dextran-induced toxicity typically results in excessive iron accumulation in organs such as the liver, heart, and kidneys, leading to tissue damage and dysfunction as seen in the Iron overload group. The observed reduction in iron deposition in major organs aftertreatment with DFO, CA-Spermine (CA-Sp), and CA-Histidine (CA-His) combinations underscores their potential efficacy in mitigating iron overload which was noticed byPrussian blue staining.

[0095] Figure 8 showed Treatment with CA-Sp and CA-His combinations reduced iron- induced organ toxicities negating effects due to Iron dextran: (A) Creatinine, (B) AST, and(C) ALT levels in the serum.

[0096] Elevated levels of Creatinine, ALT, and AST biomarkers typically indicate renal and hepatic dysfunction, often a consequence of iron overload, which can cause oxidative stress and tissue injury. The observed decrease in these markers indicates that CA-Sp and CA-His effectively alleviate the toxic effects of Iron dextran, likely by reducing iron accumulation and associated oxidative damage in vital organs such as the kidneys and liver. This outcome is comparable to the effects of established iron chelators like Deferoxamine (DFO), supporting the potential of CA-Sp and CA-His as alternative or supplementary therapies for managing iron-induced toxicity.

[0097] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.ADVANTAGES OF THE PRESENT INVENTION

[0098] The CA-biogenic amine complex offers specific binding properties, allowing targeted iron chelation.

[0099] High affinity and stability of the complexes ensure effective removal of excess iron from tissues and circulation.

[0100] These formulations are highly cost-effective, non-hazardous, natural product- derived and biocompatible, thereby minimizing adverse effects.

[0101] Using natural compounds for ICT offers several potential benefits compared to synthetic chelating agents. Some of the natural compounds explored in the present disclosure for their iron-chelating properties include the acid-base complexes of Caffeic acid (CA) with biogenic amines such as Spermine (Sp), Histidine (His), etc. The benefits of using these complexes include reduced side effects, good anti-oxidant properties, improved patient compliance, cost-effectiveness, and synergistic effects.

[0102] Thus, there is great potential for creating new water-soluble formulations of natural polyphenols without covalent bond modifications. These polyphenols' original properties can be preserved by electrostatically conjugating them with natural amines. This method can improve their biocompatibility, increasing activity and decreasing toxicity.

[0103] Using natural chemicals in ICT provides benefits across different sectors. In healthcare, it offers safer and more effective treatments for various iron overload-related illnesses. This breakthrough can benefit the pharmaceutical and biotechnology sectors regarding medication development and personalized therapy. Also, public health programs can use natural chelators to encourage preventative care and improve general population health. This breakthrough's vast use and potential influence show its significance and promise for the future.

Claims

We Claim:

1. A formulation for iron chelation therapy comprising: a water-soluble polyphenol-biogenic amine complex comprising of a polyphenol and an amine, and a 0.9% of sodium chloride; optionally one or more additive, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

2. The formulation as claimed in claim 1, wherein the water-soluble polyphenol-biogenic amine complex is present in the formulation in the range of 0.01 to 10%w / w.

3. The formulation as claimed in claim 1, wherein the polyphenol is selected from a group consisting of caffeic acid, tannic acid, gallic acid, ferulic acid, and combination thereof.

4. The formulation as claimed in claim 1, wherein the polyphenol is present in an amount in the range of 0.005 to 5%w / w.

5. The formulation as claimed in claim 1, wherein the amine is selected from a group consisting of spermine, histidine, spermidine, arginine, dopamine and combination thereof.

6. The formulation as claimed in claim 1, wherein the amine is present in an amount in the range of 0.005 to 5%w / w.

7. The formulation as claimed in claim 1, wherein the additive is selected from a group consisting of thickening agent, bulking agent, lubricant, solubilizer, binder, colouring agent, tonicity agent, buffering agent, preservative, moisturizing agent, film former / waterproofing agent, rheology modifying agent, fragrance, emollient, humectants, emulsifier, diluents, solvent and combination thereof.

8. The formulation as claimed in claim 1, wherein the additive is present in the formulation in an amount in the range of 0.1 to 10 % w / w.

9. The formulation as claimed in claim 1, wherein the formulation is administered by parentally with a dose in the range of 1-500 mg / kg / day.

10. A method of preparation of a formulation for iron chelation therapy comprising: a) adding 0.005 to 5% w / w of a polyphenol and 0.005 to 5% w / w of an amine followed by adjusting to pH-7 by adding HCI / NaHCOs to form a mixture; and b) mixing the mixture of step a) with suitable proportion in 0.9% sodium chloride and optionally one or more additive to form a formulation for iron chelation therapy, wherein water-soluble polyphenol-biogenic amine complex effectively chelates the excess iron and enable its elimination.

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